Persistence of Listeria monocytogenes in response to a natural antimicrobial, nisin : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Microbiology at Massey University, Palmerston North, New Zealand
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Date
2019
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Massey University
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Abstract
The persistence of bacteria can be defined by prolonged existence of a proportion of a population in an environment despite antimicrobial treatment. Food contamination by Listeria monocytogenes (L. monocytogenes) may be due to the persistence of L. monocytogenes despite antimicrobial treatments aimed at controlling this pathogen. How L. monocytogenes persists and survives in food environments is poorly understood.
The persistence of cells exposed to antibiotics is well recorded however the persistence following treatment with natural antimicrobials like the bacteriocin nisin (used for controlling food pathogens in a variety of food systems across a wide pH range) has not been determined. This study used two L. monocytogenes isolates, one from food and one from an animal origin, to optimize methods for obtaining L. monocytogenes persisters following nisin treatment. L. monocytogenes were treated with nisin, and a biphasic killing pattern was observed for both strains. Persister cells formed a population of cells showing tolerance to the high concentrations of nisin and proved to be non-resistant when regrown and re-exposed to nisin.
Next, this study examined persistent subpopulations of L. monocytogenes following exposure to nisin treatment under different environmental conditions (such as rich/poor media and variations in pH) representing different food systems. The pH and nutrient levels influenced the production of persister cells of L. monocytogenes. Stationary phase cells re-suspended in a nutrient rich environment (Trypticase Soy broth, TSB) showed greater survival than in a nutrient limiting environment (spent TSB medium) under nisin treatment. However, traditional microbiological techniques such as agar plating are limited in providing information of persister cells exposed to antimicrobial substances.
Flow cytometry has potential to identify persister cells that although they cannot be cultured, can still pose a threat to human health. The L. monocytogenes stationary phase populations in spent medium or re-suspended in fresh TSB medium were individually treated with nisin, and the treated cells were assayed using flow cytometry combined with live/dead staining. The results revealed a reduction in viable populations and the differentiation of living cells and dead/damaged cells within the treated population. This finding was consistent with previous results from plate count experiments. Distinct changes in cell permeability were detected within 2hrs of the nisin treatment in spent medium and re-suspended in fresh medium, indicating possible variations in the mechanisms of persister formation between the two groups. The persistence of a L.monocytogenes stationary phase population when facing nisin treatment was investigated for gene expression with RNA extracts obtained after 90mins of nisin treatment. RNA Seq analysis was used for gene expression profiling of the persister cells in spent (persister N) and rich medium (persister TN) compared with untreated cells. Functional genes associated with the persister populations were identified in multiple systems including heat shock related stress response, cell wall synthesis, ATP-binding cassette (ABC) transport system, phosphotransferase system (PTS system), and SOS/DNA repair. Differences were found in gene regulation between persister N and persister TN populations. Nutrition may be associated with the variations in gene expression resulting in variations in the size or composition of the persistent populations. This study provided information on the formation of persister cells exposed to nisin and provides some insight into possible mechanisms of impeding bacterial persistence.
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Listed in 2019 Dean's List of Exceptional Theses
Figure 2.1 re-used with publisher's permission.
Figure 2.1 re-used with publisher's permission.
Keywords
Listeria monocytogenes, Genetics, Nisin, Drug resistance in microorganisms, Food, Microbiology, Dean's List of Exceptional Theses